Enzyme-linked Immunosorbent Assay (elisa )

Introduction to ELISA

Enzyme-linked Immunosorbent Assay (ELISA) is a test that has evolved from other types of immunoassays since the early 1970s. It is now one of the most advanced and widely used laboratory techniques in clinical and translational medicine. This assay is crucial for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. With this test, it is possible to detect and measure antibodies in your blood, which can determine if you have antibodies related to certain infectious conditions (Engvall & Perlmann, 1971; Voller et al., 1976).

Applications of ELISA

ELISA can be applied to the determination of serum antibody concentrations in virus tests. ELISA tests are also found in home pregnancy tests and the food industry, where they detect potential food allergens such as milk, peanuts, walnuts, almonds, and eggs (Lequin, 2005). In toxicology, ELISA serves as a rapid presumptive screen for certain classes of drugs. The ELISA technique is widely used in various clinical and medical areas such as immunology, biological pharmacy, and the diagnostic industry. It is employed for the detection of antibodies in blood samples to determine past exposure to diseases, such as lung diseases, trichinosis, HIV, and bird flu (Crowther, 2000).

Principle of ELISA

Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs provide a useful measurement of antigen or antibody concentration. A significant aspect of this application is that ELISA can utilize antibodies to recognize antigens, making it a sensitive immunoassay for detecting specific proteins, especially antibodies or antigens (Engvall & Perlmann, 1971).

ELISA involves the detection of an "analyte" in a liquid sample using liquid reagents or dry strips. In dry analysis, strips can be used in reflectometry. Quantitative readings are usually based on the detection of the intensity of transmitted light by spectrophotometry of a specific wavelength. The sensitivity of detection depends on the amplification of the signal during the analytic reaction. In some enzymatic reactions, the signal generated by the enzyme is linked to the detection reagents in fixed proportions, allowing accurate quantification (Voller et al., 1976).

Types of ELISA

• Direct ELISA: Suitable for detecting proteinaceous antigens and may require pre-purification of samples. It is performed when the desired antibody is available in a pre-conjugated state.
• Indirect ELISA: When the primary antibody is not conjugated, indirect ELISA is required, where a conjugated secondary antibody targets the isotope of the primary antibody.
• Sandwich ELISA: Quantifies the measure of antigen between two layers of antibodies. The antigen must contain no less than two antigenic sites capable of binding to an antibody, as at least two antibodies act in a sandwich format.
• Competitive ELISA: In this type, the antibody is initially incubated in solution with a specimen containing antigen. The antigen-antibody mixture is then added to the microtiter well coated with antigen. The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. After washing the well, an enzyme-conjugated secondary antibody specific for the isotope of the primary antibody is added to determine the amount of primary antibody bound to the well (Crowther, 2000).

Specific Applications of ELISA

An ELISA for the specific detection of RHDV2 antigen has been designed and validated. Some specific serological tools are used for monitoring virus circulation and controlling diseases for the detection of RHDV2 antigens in rabbit liver homogenates. This test, based on the use of RHDV2 monoclonal antibodies and anti-RHDV2 goat polyclonal antibodies, has high sensitivity and successfully detects RHDV2 and RHDV2 recombinant virions. Rabbit haemorrhagic disease virus (RHDV) is a prototype virus of the lagovirus genus, found in many other countries in both wild and domestic rabbits. ELISA methods are used for the veterinary diagnosis of RHD in domestic rabbits, which has been developed and well-characterized. This ELISA is used for detecting RHDV2 and serves as a specific virological tool for monitoring virus circulation, allowing better control of these diseases (OIE, 2018).

Saxitoxin (STX) is a marine toxin that causes paralytic shellfish poisoning, showing morbidity and mortality in humans. ELISA is used for the quantitative detection of Saxitoxin in human blood using synthetic blood calibrators, which can sometimes be inaccurate. The mouse bioassay is a common method that measures the presence of active toxin by injecting mice with extracts from shellfish and monitoring the mortality rate. In this study, indirect competitive ELISA is utilized for polyclonal antibodies that recognize STX for detection. This ELISA significantly cross-reacts with gonyautoxins 2 and 3. ELISA has the ability to detect pattern recognition of epitopes and multiple toxin analogues or derivatives. It is marked for qualitative or quantitative detection of STX in freshwater and brackish water samples (Usleber et al., 2001).

Conclusion

ELISA is a versatile and powerful tool in both clinical and research settings. Its applications span a wide range of fields, from medical diagnostics to food safety and environmental monitoring. The ongoing development and refinement of ELISA methodologies continue to expand its capabilities, making it an indispensable technique in modern science and medicine.

References

• Crowther, J. R. (2000). The ELISA Guidebook. Humana Press.
• Engvall, E., & Perlmann, P. (1971). Enzyme-linked immunosorbent assay (ELISA). Journal of Immunology, 109(1), 129-135.
• Lequin, R. M. (2005). Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical Chemistry, 51(12), 2415-2418.
• OIE. (2018). Rabbit Haemorrhagic Disease. In Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organisation for Animal Health.
• Usleber, E., Dietrich, R., & Märtlbauer, E. (2001). Immunochemical detection of microbial toxins. Journal of AOAC International, 84(5), 1381-1389.
• Voller, A., Bidwell, D. E., & Bartlett, A. (1976). Enzyme immunoassays in diagnostic medicine. Theory and practice. Bulletin of the World Health Organization, 53(1), 55-65.